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Gene expression profiling was performed on 97 cases of infant ALL from Children's Oncology Group Trial P9407. Statistical modeling of an outcome predictor revealed 3 genes highly predictive of event-free survival (EFS), beyond age and MLL status: FLT3, IRX2, and TACC2. Low FLT3 expression was found in a group of infants with excellent outcome (n = 11; 5-year EFS of 100%), whereas differential expression of IRX2 and TACC2 partitioned the remaining infants into 2 groups with significantly different survivals (5-year EFS of 16% vs 64%; P < .001). When infants with MLL-AFF1 were analyzed separately, a 7-gene classifier was developed that split them into 2 distinct groups with significantly different outcomes (5-year EFS of 20% vs 65%; P < .001). In this classifier, elevated expression of NEGR1 was associated with better EFS, whereas IRX2, EPS8, and TPD52 expression were correlated with worse outcome. This classifier also predicted EFS in an independent infant ALL cohort from the Interfant-99 trial. When evaluating expression profiles as a continuous variable relative to patient age, we further identified striking differences in profiles in infants less than or equal to 90 days of age and those more than 90 days of age. These age-related patterns suggest different mechanisms of leukemogenesis and may underlie the differential outcomes historically seen in these age groups.

IDH1 SNP rs11554137 was recently reported in association with poor prognosis in normal karyotype adult acute myeloid leukemia (AML). We aimed to determine the prevalence, clinical associations, and prognostic significance of SNP rs11554137 in unselected pediatric and adult AML patients. Diagnostic marrow specimens from 527 AML patients treated on the pediatric trial Children's Oncology Group-AAML03P1 (N = 253) or adult SWOG trials (N = 274) were analyzed for the presence of the SNP. SNP rs11554137 was present in 11% of all patients. SNP status had no prognostic impact on survival in pediatric patients. In adult AML, overall survival for SNP-positive patients was 10% versus 18% for SNP-negative patients (P = .44). Among the 142 adults who achieved complete remission, 5-year relapse-free survival was significantly worse for SNP-positive patients (0% vs 25%, P = .0014). However, among adults with normal cytogenetics, FLT3/ITD was present in 90% of SNP-positive patients versus 59% of SNP-negative patients (P = .0053). In multivariate analysis, adjusting for the effects of age, cytogenetics, and FLT3/ITD, the independent prognostic effect of SNP positivity was not statistically significant (hazard ratio = 1.72, P = .18). The clinical profile of SNP-positive patients suggests that SNP rs11554137 may have biologic effects that bear further investigation. The clinical trials in this study are registered at http://www.clinicaltrials.gov as #NCT000707174 and #NCT00899171.

Background: The augmented BFM regimen improves outcome for children with NCI high acute lymphoblastic leukemia (ALL). Patient age, sex, and presenting white blood cell count (WBC) can be used to identify a subset of approximately 12% of children with B-precursor ALL that had a 5-year continuous complete remission (CCR) rate of only about 50% on earlier Pediatric Oncology Group (POG) trials., Procedures: Children's Oncology Group trial P9906 evaluated a modified augmented BFM regimen in 267 patients with particularly high risk B-precursor ALL. Minimal residual disease (MRD) was assessed in blood at day 8 and in marrow at day 29 of induction and correlated with outcome., Results: The 5-year CCR probability for patients in P9906 was significantly better than that observed for similar patients on POG trials 8602/9006 (62.2 ± 3.7% vs. 50.6 ± 2.4%; P = 0.0007) but similar to POG 9406 (63.5 ± 2.4%; P = 0.81). Interim analysis showed poor central nervous system (CNS) control, especially in patients with initial WBC ≥ 100,000/microliter. Day 29 marrow MRD positive (≥ 0.01%) vs. negative patients had 5 year CCR rates of 37.1 ± 7.4% vs. 72.6 ± 4.3%; day 8 blood MRD positive vs. negative patients had 5 year CCR rates of 57.1 ± 4.6% vs.83.6 ± 6.3%. End induction marrow MRD predicted marrow but not CNS relapse. In multivariate analysis, day 29 MRD > 0.01%, initial WBC ≥ 100,000/µl, male gender, and day 8 blood MRD > 0.01% were significant prognostic factors., Conclusions: Augmented BFM therapy improved outcome for children with higher risk ALL. Day 8 blood and day 29 marrow MRD were strong prognostic factors in these patients., (Copyright © 2011 Wiley-Liss, Inc.)

We sequenced 120 candidate genes in 187 high-risk childhood B-precursor acute lymphoblastic leukemias, the largest pediatric cancer genome sequencing effort reported to date. Integrated analysis of 179 validated somatic sequence mutations with genome-wide copy number alterations and gene expression profiles revealed a high frequency of recurrent somatic alterations in key signaling pathways, including B-cell development/differentiation (68% of cases), the TP53/RB tumor suppressor pathway (54%), Ras signaling (50%), and Janus kinases (11%). Recurrent mutations were also found in ETV6 (6 cases), TBL1XR1 (3), CREBBP (3), MUC4 (2), ASMTL (2), and ADARB2 (2). The frequency of mutations within the 4 major pathways varied markedly across genetic subtypes. Among 23 leukemias expressing a BCR-ABL1-like gene expression profile, 96% had somatic alterations in B-cell development/differentiation, 57% in JAK, and 52% in both pathways, whereas only 9% had Ras pathway mutations. In contrast, 21 cases defined by a distinct gene expression profile coupled with focal ERG deletion rarely had B-cell development/differentiation or JAK kinase alterations but had a high frequency (62%) of Ras signaling pathway mutations. These data extend the range of genes that are recurrently mutated in high-risk childhood B-precursor acute lymphoblastic leukemia and highlight important new therapeutic targets for selected patient subsets.

Virus-like particles (VLPs) of bacteriophage MS2 possess numerous features that make them well-suited for use in targeted delivery of therapeutic and imaging agents. MS2 VLPs can be rapidly produced in large quantities using in vivo or in vitro synthesis techniques. Their capsids can be modified in precise locations via genetic insertion or chemical conjugation, facilitating the multivalent display of targeting ligands. MS2 VLPs also self-assemble in the presence of nucleic acids to specifically encapsidate siRNA and RNA-modified cargos. Here we report the use of MS2 VLPs to selectively deliver nanoparticles, chemotherapeutic drugs, siRNA cocktails, and protein toxins to human hepatocellular carcinoma (HCC). MS2 VLPs modified with a peptide (SP94) that binds HCC exhibit a 10(4)-fold higher avidity for HCC than for hepatocytes, endothelial cells, monocytes, or lymphocytes and can deliver high concentrations of encapsidated cargo to the cytosol of HCC cells. SP94-targeted VLPs loaded with doxorubicin, cisplatin, and 5-fluorouracil selectively kill the HCC cell line, Hep3B, at drug concentrations <1 nM, while SP94-targeted VLPs that encapsidate a siRNA cocktail, which silences expression of cyclin family members, induce growth arrest and apoptosis of Hep3B at siRNA concentrations <150 pM. Impressively, MS2 VLPs, when loaded with ricin toxin A-chain (RTA) and modified to codisplay the SP94 targeting peptide and a histidine-rich fusogenic peptide (H5WYG) that promotes endosomal escape, kill virtually the entire population of Hep3B cells at an RTA concentration of 100 fM without affecting the viability of control cells. Our results demonstrate that MS2 VLPs, because of their tolerance of multivalent peptide display and their ability to specifically encapsidate a variety of chemically disparate cargos, induce selective cytotoxicity of cancer in vitro and represent a significant improvement in the characteristics of VLP-based delivery systems.

Older acute myeloid leukemia (AML) patients with a chromosome 5q deletion have poor outcomes with conventional chemotherapy. This phase 2 study explored the safety and efficacy of single-agent lenalidomide in previously untreated older AML patients with del(5q) who declined standard chemotherapy. Patients were treated with lenalidomide 50 mg daily for 28 days as induction therapy and 10 mg daily for 21 days of a 28-day cycle as maintenance until disease progression or unacceptable toxicity. Among 37 evaluable patients, the median age was 74 years (range, 60-94), 21 (57%) were female, 19 (51%) had prior myelodysplastic syndrome, and 30 (81%) had pretreatment cytogenetic studies evaluated centrally. Six had isolated del(5q), 1 had del(5q) and +8, 23 had complex cytogenetics, and 7 others had del(5q) identified locally. Fourteen patients (38%) completed induction therapy: 7 patients died during induction therapy, 8 had disease progression, 7 had nonfatal adverse events, and 1 entered hospice. Eight patients started maintenance therapy. Five patients (14%) achieved a partial or complete response, 2 with isolated del(5q) and 3 with complex cytogenetics. Relapse-free survival was 5 months (range, 0-19). Median overall survival was 2 months for the entire population. In conclusion, lenalidomide as a single agent has modest activity in older del(5q) AML patients. Southwest Oncology Group Study S0605 is registered at www.clinicaltrials.gov as NCT00352365.

Gamma secretase inhibitors (GSIs) comprise a growing class of compounds that interfere with the membrane-bound Notch signaling protein and its downstream intra-nuclear transcriptional targets. As GSI-I (Z-LLNle-CHO) is also a derivative of a widely used proteosome inhibitor MG-132, we hypothesized that this compound might be active in precursor-B acute lymphoblastic leukemia (ALL) cell lines and patient samples. We found that GSI-I treatment of precursor-B ALL blasts induced apoptotic cell death within 18-24 h. With confirmation using RNA and protein analyses, GSI-I blocked nuclear accumulation of cleaved Notch1 and Notch2, and inhibited Notch targets Hey2 and Myc. Microarray analyses of 207 children with high-risk precursor-B ALL demonstrate that Notch pathway expression is a common feature of these neoplasms. However, microarray studies also implicated additional transcriptional targets in GSI-I-dependent cell death, including genes in the unfolded protein response, nuclear factor-κB and p53 pathways. Z-LLNle-CHO blocks both γ-secretase and proteosome activity, inducing more robust cell death in precursor-B ALL cells than either proteosome-selective or γ-secretase-selective inhibitors alone. Using Z-LLNle-CHO in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) precursor-B ALL xenograft model, we found that GSI-I alone delayed or prevented engraftment of B-lymphoblasts in 50% of the animals comprising the experimental group, suggesting that this compound is worthy of additional testing.

Despite impressive advances in cure rates for childhood acute lymphoblastic leukemia (ALL), ALL remains the leading cause of disease-related death in young people and new therapeutic approaches directed against rational therapeutic targets are urgently required to improve treatment outcomes. This is particularly true for ALL in older children, adolescents, and adults, in whom treatment outcomes are markedly inferior to those of young children. A major goal of current leukemia research is to use comprehensive genomic analysis of the leukemic cell genome, transcriptome, and epigenome to identify critical new genomic alterations that drive leukemogenesis and influence responsiveness to therapy. Genomic analyses in childhood ALL have been remarkably informative and have identified a number of new structural genetic alterations that play important roles in the establishment of the leukemic clone and determine risk of relapse. Notably, many high-risk ALL cases harbor loss-of-function and dominant mutations of genes that encode transcriptional regulators of lymphoid development coupled with mutations that result in activation of cytokine receptor and kinase signaling pathways. These advances have resulted in new diagnostic approaches and therapeutic trials in ALL. This review will discuss these advances and outline challenges for future studies, including the potential role of genome-wide sequencing approaches and the need for detailed studies of the genetics of ALL in the adolescent and young adult population.

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes.

Although five-year survival rates for childhood acute lymphoblastic leukemia (ALL) are now over 80% in most industrialized countries, not all children have benefited equally from this progress. Ethnic differences in survival after childhood ALL have been reported in many clinical studies, with poorer survival observed among African Americans or those with Hispanic ethnicity when compared with European Americans or Asians. The causes of ethnic differences remain uncertain, although both genetic and non-genetic factors are likely important. Interrogating genome-wide germline SNP genotypes in an unselected large cohort of children with ALL, we observed that the component of genomic variation that co-segregated with Native American ancestry was associated with risk of relapse (P = 0.0029) even after adjusting for known prognostic factors (P = 0.017). Ancestry-related differences in relapse risk were abrogated by the addition of a single extra phase of chemotherapy, indicating that modifications to therapy can mitigate the ancestry-related risk of relapse.

Purpose: Although the majority of children with acute lymphoblastic leukemia (ALL) are cured with current therapy, the event-free survival (EFS) of infants with ALL, particularly those with mixed lineage leukemia (MLL) gene rearrangements, is only 30% to 40%. Relapse has been the major source of treatment failure for these patients. The parallel Children's Cancer Group (CCG) 1953 and Pediatric Oncology Group (POG) 9407 studies were designed to test the hypothesis that more intensive therapy, including dose intensification of chemotherapy, and hematopoietic stem-cell transplantation (HSCT) would improve the outcome for this group of patients., Patients and Methods: One hundred eighty-nine infants (CCG 1953, n = 115; POG 9407, n = 74) were enrolled between October 1996 and August 2000. For infants with the MLL gene rearrangement and an appropriate donor, HSCT was the preferred treatment on CCG 1953 and investigator option on POG 9407 after completion of the second phase of therapy. Fifty-three infants underwent HSCT., Results: The 5-year EFS rate was 48.8% (95% CI, 33.9% to 63.7%) in patients who received HSCT and 48.7% (95% CI, 33.8% to 63.6%) in patients treated with chemotherapy alone (P = .60). Transplantation outcomes were not affected by the preparatory regimen or donor source., Conclusion: Our data suggest that routine use of HSCT for infants with MLL-rearranged ALL is not indicated. However, limited by small numbers, this study should not be considered the definitive answer to this question.

Few studies have examined the outcome of large numbers of patients with the microgranular variant (M3V) of acute promyelocytic leukemia (APL) in the all-trans retinoic acid era. Here, the outcome of 155 patients treated with all-trans retinoic acid-based therapy on 3 clinical trials, North American Intergroup protocol I0129 and Programa para el Estudio de la Terapéutica en Hemopatía Maligna protocols LPA96 and LPA99, are reported. The complete remission rate for all 155 patients was 82%, compared with 89% for 748 patients with classical M3 disease. The incidence of the APL differentiation syndrome was 26%, compared with 25% for classical M3 patients, and the early death rate was 13.6% compared with 8.4% for patients with classical M3 morphology. With a median follow-up time among survivors of 7.6 years (range 3.6-14.5), the 5-year overall survival, disease-free survival, and cumulative incidence of relapse for patients with M3V were 70%, 73%, and 24%, respectively. With a median follow-up time among survivors of 7.6 years (range 0.6-14.3), the 5-year overall survival, disease-free survival, and cumulative incidence of relapse among patients with classical M3 morphology were 80% (P = .006 compared with M3V), 81% (P = .07), and 15% (P = .005), respectively. When outcomes were adjusted for the white blood cell count or the relapse risk score, none of these outcomes were significantly different between patients with M3V and classical M3 APL.

To resolve the genetic heterogeneity within pediatric high-risk B-precursor acute lymphoblastic leukemia (ALL), a clinically defined poor-risk group with few known recurring cytogenetic abnormalities, we performed gene expression profiling in a cohort of 207 uniformly treated children with high-risk ALL. Expression profiles were correlated with genome-wide DNA copy number abnormalities and clinical and outcome features. Unsupervised clustering of gene expression profiling data revealed 8 unique cluster groups within these high-risk ALL patients, 2 of which were associated with known chromosomal translocations (t(1;19)(TCF3-PBX1) or MLL), and 6 of which lacked any previously known cytogenetic lesion. One unique cluster was characterized by high expression of distinct outlier genes AGAP1, CCNJ, CHST2/7, CLEC12A/B, and PTPRM; ERG DNA deletions; and 4-year relapse-free survival of 94.7% ± 5.1%, compared with 63.5% ± 3.7% for the cohort (P = .01). A second cluster, characterized by high expression of BMPR1B, CRLF2, GPR110, and MUC4; frequent deletion of EBF1, IKZF1, RAG1-2, and IL3RA-CSF2RA; JAK mutations and CRLF2 rearrangements (P < .0001); and Hispanic ethnicity (P < .001) had a very poor 4-year relapse-free survival (21.0% ± 9.5%; P < .001). These studies reveal striking clinical and genetic heterogeneity in high-risk ALL and point to novel genes that may serve as new targets for diagnosis, risk classification, and therapy.

Arsenic trioxide (As(2)O(3)) is a highly effective treatment for patients with relapsed acute promyelocytic leukemia (APL); its role as consolidation treatment for patients in first remission has not been defined. We randomized 481 patients (age ≥ 15 years) with untreated APL to either a standard induction regimen of tretinoin, cytarabine, and daunorubicin, followed by 2 courses of consolidation therapy with tretinoin plus daunorubicin, or to the same induction and consolidation regimen plus two 25-day courses of As(2)O(3) consolidation immediately after induction. After consolidation, patients were randomly assigned to one year of maintenance therapy with either tretinoin alone or in combination with methotrexate and mercaptopurine. Ninety percent of patients on each arm achieved remission and were eligible to receive their assigned consolidation therapy. Event-free survival, the primary end point, was significantly better for patients assigned to receive As(2)O(3) consolidation, 80% compared with 63% at 3 years (stratified log-rank test, P < .0001). Survival, a secondary end point, was better in the As(2)O(3) arm, 86% compared with 81% at 3 years (P = .059). Disease-free survival, a secondary end point, was significantly better in the As(2)O(3) arm, 90% compared with 70% at 3 years (P < .0001). The addition of As(2)O(3) consolidation to standard induction and consolidation therapy significantly improves event-free and disease-free survival in adults with newly diagnosed APL. This trial was registered at clinicaltrials.gov (NCT00003934).

Children with Down syndrome (DS) have an increased risk of acute lymphoblastic leukemia (ALL) and an inferior outcome. We reviewed data from 2811 children with ALL enrolled in Children's Oncology Group P9900, which included prospective testing for the major cytogenetic lesions in childhood ALL: ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL translocations and trisomies of chromosomes 4 and 10. Eighty (3%) B-precursor ALL patients had DS. Age, sex, white blood cell count, and risk group were similar between DS-ALL and non-DS-ALL but significantly more patients with DS-ALL were white (91.2% vs 76.4%, P = .001). Children with DS-ALL had lower rates of the favorable cytogenetic lesions ETV6-RUNX1 (2.5% vs 24%, P < .001) and trisomies 4 and 10 (7.7% vs 24%, P < .001). Five-year event-free (EFS) and overall survival (OS) were inferior in children with DS-ALL: 69.9% +/- 8.6% versus 78.1% +/- 1.2% (P = .078), and 85.8% +/- 6.5% versus 90.0% +/- 0.9% (P = .033). However, when children with MLL translocations, BCR-ABL1, ETV6-RUNX1, and trisomies 4 and 10 were excluded, the EFS and OS were similar for children with and without DS (EFS 68.0 %+/- 9.3% vs 70.5% +/- 1.9%, P = .817; and OS 86.7% +/- 6.7% vs 85.4% +/- 1.5%; P = .852), both overall and adjusted for race. DS-ALL displays a unique spectrum of biologic subtypes with different frequencies of sentinel cytogenetic lesions having a large influence on outcome.

Background: Imatinib is a highly effective treatment for chronic myeloid leukemia. It was approved by the Food and Drug Administration in 2001 and thereafter rapidly became front-line therapy. This study characterized the prevailing chronic myeloid leukemia therapies in the United States and assessed the impact of imatinib on chronic myeloid leukemia survival and mortality rates in the general population., Methods: Investigators with the National Cancer Institute's Patterns of Care study reviewed medical records and queried physicians regarding therapy for 423 patients with chronic myeloid leukemia diagnosed in 2003 who were randomly selected from cancer registries in the Surveillance, Epidemiology, and End Results Program. Characteristics associated with the receipt of imatinib were documented, as were survival differences between those who received imatinib and those who did not. Population-based data were used to assess chronic myeloid leukemia survival and mortality rates in time periods before and after the introduction of imatinib., Results: Imatinib was administered to 76% of patients in the Patterns of Care study. Imatinib use was inversely associated with age: 90%, 75%, and 46% for patients ages 20 to 59 years, 60 to 79 years, and 80 or more years, respectively. Elderly patients who received imatinib survived significantly longer than those who did not. After adjusting for age, imatinib use did not vary significantly by race/ethnicity, socioeconomic status, urban/rural residence, presence of comorbid conditions, or insurance status. Overall, chronic myeloid leukemia survival in the Surveillance, Epidemiology, and End Results population improved, and mortality in the United States declined dramatically during the period when imatinib became widely available; these improvements diminished with increasing age., Conclusion: Age disparities in treatment with imatinib likely contributed to worse survival for many elderly patients with chronic myeloid leukemia., (Copyright 2010 Elsevier Inc. All rights reserved.)

Gene expression profiling of 207 uniformly treated children with high-risk B-progenitor acute lymphoblastic leukemia revealed 29 of 207 cases (14%) with markedly elevated expression of CRLF2 (cytokine receptor-like factor 2). Each of the 29 cases harbored a genomic rearrangement of CRLF2: 18 of 29 (62%) had a translocation of the immunoglobulin heavy chain gene IGH@ on 14q32 to CRLF2 in the pseudoautosomal region 1 of Xp22.3/Yp11.3, whereas 10 (34%) cases had a 320-kb interstitial deletion centromeric of CRLF2, resulting in a P2RY8-CRLF2 fusion. One case had both IGH@-CRLF2 and P2RY8-CRLF2, and another had a novel CRLF2 rearrangement. Only 2 of 29 cases were Down syndrome. CRLF2 rearrangements were significantly associated with activating mutations of JAK1 or JAK2, deletion or mutation of IKZF1, and Hispanic/Latino ethnicity (Fisher exact test, P < .001 for each). Within this cohort, patients with CRLF2 rearrangements had extremely poor treatment outcomes compared with those without CRLF2 rearrangements (35.3% vs 71.3% relapse-free survival at 4 years; P < .001). Together, these observations suggest that activation of CRLF2 expression, mutation of JAK kinases, and alterations of IKZF1 cooperate to promote B-cell leukemogenesis and identify these pathways as important therapeutic targets in this disease.

Recent whole-genome sequencing efforts led to the identification of IDH1(R132) mutations in acute myeloid leukemia (AML) patients. We studied the prevalence and clinical implications of IDH1 genomic alterations in pediatric and adult AML. Diagnostic DNA from 531 AML patients treated on Children's Oncology Group trial COG-AAML03P1 (N=257), and Southwest Oncology Group trials SWOG-9031, SWOG-9333 and SWOG-9500 (N=274), were tested for IDH1 mutations. Codon R132 mutations were absent in the pediatric cohort, but were found in 12 of 274 adult patients (4.4%, 95% CI 2.3-7.5). IDH1(R132) mutations occurred most commonly in patients with normal karyotype, and those with FLT3/ITD and NPMc mutations. Patients with IDH1(R132) mutations trended toward higher median diagnostic white blood cell counts (59.2 x 10(9) vs 29.1 x 10(9) per liter, P=0.19) than those without mutations, but the two groups did not differ significantly in age, bone marrow blast percentage, overall survival or relapse-free survival. Eleven patients (2.1%) harbored a novel V71I sequence alteration, which was found to be a germ-line polymorphism. IDH1 mutations were not detected in pediatric AML, and are uncommon in adult AML.

To determine whether gene expression profiling could improve outcome prediction in children with acute lymphoblastic leukemia (ALL) at high risk for relapse, we profiled pretreatment leukemic cells in 207 uniformly treated children with high-risk B-precursor ALL. A 38-gene expression classifier predictive of relapse-free survival (RFS) could distinguish 2 groups with differing relapse risks: low (4-year RFS, 81%, n = 109) versus high (4-year RFS, 50%, n = 98; P < .001). In multivariate analysis, the gene expression classifier (P = .001) and flow cytometric measures of minimal residual disease (MRD; P = .001) each provided independent prognostic information. Together, they could be used to classify children with high-risk ALL into low- (87% RFS), intermediate- (62% RFS), or high- (29% RFS) risk groups (P < .001). A 21-gene expression classifier predictive of end-induction MRD effectively substituted for flow MRD, yielding a combined classifier that could distinguish these 3 risk groups at diagnosis (P < .001). These classifiers were further validated on an independent high-risk ALL cohort (P = .006) and retainedindependent prognostic significance (P < .001) in the presence of other recently described poor prognostic factors (IKAROS/IKZF1 deletions, JAK mutations, and kinase expression signatures). Thus, gene expression classifiers improve ALL risk classification and allow prospective identification of children who respond or fail current treatment regimens. These trials were registered at http://clinicaltrials.gov under NCT00005603.

Attempts to overcome multi-drug resistance in acute myeloid leukaemia (AML) have been limited by toxicities. To investigate the effect of reducing peak drug levels, we performed sequential phase II studies using continuous infusion daunorubicin and cytarabine without (AD) and then with ciclosporin (ADC) in older patients with AML. Untreated patients (age 56+ years) received daunorubicin (45 mg/m2 per day for 3 d) and cytarabine (200 mg/m2 per day for 7 d), both by continuous infusion, without (S0112, 60 patients) and then with (S0301, 50 patients) the addition of ciclosporin. Complete response (CR) rates were 38% on S0112 and 44% on S0301. Fatal induction toxicities occurred in 17% and 12% respectively, arising primarily from infection and haemorrhage. Median overall and relapse-free survival was 7 and 8 months for AD respectively, and 6 and 14 months for ADC. Patients with phenotypic or functional P-glycoprotein had somewhat higher CR rates with ADC than AD, although confidence intervals overlapped. In these sequential trials, continuous infusion AD produced CR rates comparable to those with bolus daunorubicin. The addition of ciclosporin did not cause undue toxicities, produced a similar CR rate, and possibly improved relapse-free survival. Further correlate analyses did not identify a subpopulation specifically benefitting from the addition of ciclosporin.

Aneuploidy and translocations are hallmarks of B-progenitor acute lymphoblastic leukemia (ALL), but many individuals with this cancer lack recurring chromosomal alterations. Here we report a recurring interstitial deletion of the pseudoautosomal region 1 of chromosomes X and Y in B-progenitor ALL that juxtaposes the first, noncoding exon of P2RY8 with the coding region of CRLF2. We identified the P2RY8-CRLF2 fusion in 7% of individuals with B-progenitor ALL and 53% of individuals with ALL associated with Down syndrome. CRLF2 alteration was associated with activating JAK mutations, and expression of human P2RY8-CRLF2 together with mutated mouse Jak2 resulted in constitutive Jak-Stat activation and cytokine-independent growth of Ba/F3 cells overexpressing interleukin-7 receptor alpha. Our findings indicate that these two genetic lesions together contribute to leukemogenesis in B-progenitor ALL.

After DNA replication, sister chromatids must be untangled, or decatenated, before mitosis so that chromatids do not tear during anaphase. Topoisomerase IIalpha (Topo IIalpha) is the major decatenating enzyme. Topo IIalpha inhibitors prevent decatenation, causing cells to arrest during mitosis. Here we report that acute myeloid leukemia cells fail to arrest at the mitotic decatenation checkpoint, and their progression through this checkpoint is regulated by the DNA repair component Metnase (also termed SETMAR). Metnase contains a SET histone methylase and transposase nuclease domain, and is a component of the nonhomologous end-joining DNA double-strand break repair pathway. Metnase interacts with Topo IIalpha and enhances its decatenation activity. Here we show that multiple types of acute leukemia cells have an attenuated mitotic arrest when decatenation is inhibited and that in an acute myeloid leukemia (AML) cell line this is mediated by Metnase. Of further importance, Metnase permits continued proliferation of these AML cells even in the presence of the clinical Topo IIalpha inhibitor VP-16. In vitro, purified Metnase prevents VP-16 inhibition of Topo IIalpha decatenation of tangled DNA. Thus, Metnase expression levels may predict AML resistance to Topo IIalpha inhibitors, and Metnase is a potential therapeutic target for small molecule interference.

Pediatric acute lymphoblastic leukemia (ALL) is a heterogeneous disease consisting of distinct clinical and biological subtypes that are characterized by specific chromosomal abnormalities or gene mutations. Mutation of genes encoding tyrosine kinases is uncommon in ALL, with the exception of Philadelphia chromosome-positive ALL, where the t(9,22)(q34;q11) translocation encodes the constitutively active BCR-ABL1 tyrosine kinase. We recently identified a poor prognostic subgroup of pediatric BCR-ABL1-negative ALL patients characterized by deletion of IKZF1 (encoding the lymphoid transcription factor IKAROS) and a gene expression signature similar to BCR-ABL1-positive ALL, raising the possibility of activated tyrosine kinase signaling within this leukemia subtype. Here, we report activating mutations in the Janus kinases JAK1 (n = 3), JAK2 (n = 16), and JAK3 (n = 1) in 20 (10.7%) of 187 BCR-ABL1-negative, high-risk pediatric ALL cases. The JAK1 and JAK2 mutations involved highly conserved residues in the kinase and pseudokinase domains and resulted in constitutive JAK-STAT activation and growth factor independence of Ba/F3-EpoR cells. The presence of JAK mutations was significantly associated with alteration of IKZF1 (70% of all JAK-mutated cases and 87.5% of cases with JAK2 mutations; P = 0.001) and deletion of CDKN2A/B (70% of all JAK-mutated cases and 68.9% of JAK2-mutated cases). The JAK-mutated cases had a gene expression signature similar to BCR-ABL1 pediatric ALL, and they had a poor outcome. These results suggest that inhibition of JAK signaling is a logical target for therapeutic intervention in JAK mutated ALL.

Background: Despite best current therapy, up to 20% of pediatric patients with acute lymphoblastic leukemia (ALL) have a relapse. Recent genomewide analyses have identified a high frequency of DNA copy-number abnormalities in ALL, but the prognostic implications of these abnormalities have not been defined., Methods: We studied a cohort of 221 children with high-risk B-cell-progenitor ALL with the use of single-nucleotide-polymorphism microarrays, transcriptional profiling, and resequencing of samples obtained at diagnosis. Children with known very-high-risk ALL subtypes (i.e., BCR-ABL1-positive ALL, hypodiploid ALL, and ALL in infants) were excluded from this cohort. A copy-number abnormality was identified as a predictor of poor outcome, and it was then tested in an independent validation cohort of 258 patients with B-cell-progenitor ALL., Results: More than 50 recurring copy-number abnormalities were identified, most commonly involving genes that encode regulators of B-cell development (in 66.8% of patients in the original cohort); PAX5 was involved in 31.7% and IKZF1 in 28.6% of patients. Using copy-number abnormalities, we identified a predictor of poor outcome that was validated in the independent validation cohort. This predictor was strongly associated with alteration of IKZF1, a gene that encodes the lymphoid transcription factor IKAROS. The gene-expression signature of the group of patients with a poor outcome revealed increased expression of hematopoietic stem-cell genes and reduced expression of B-cell-lineage genes, and it was similar to the signature of BCR-ABL1-positive ALL, another high-risk subtype of ALL with a high frequency of IKZF1 deletion., Conclusions: Genetic alteration of IKZF1 is associated with a very poor outcome in B-cell-progenitor ALL., (2009 Massachusetts Medical Society)

Context: Pediatric acute lymphoblastic leukemia (ALL) is the prototype for a drug-responsive malignancy. Although cure rates exceed 80%, considerable unexplained interindividual variability exists in treatment response., Objectives: To assess the contribution of inherited genetic variation to therapy response and to identify germline single-nucleotide polymorphisms (SNPs) associated with risk of minimal residual disease (MRD) after remission induction chemotherapy., Design, Setting, and Patients: Genome-wide interrogation of 476,796 germline SNPs to identify genotypes that were associated with MRD in 2 independent cohorts of children with newly diagnosed ALL: 318 patients in St Jude Total Therapy protocols XIIIB and XV and 169 patients in Children's Oncology Group trial P9906. Patients were enrolled between 1994 and 2006 and last follow-up was in 2006., Main Outcome Measures: Minimal residual disease at the end of induction therapy, measured by flow cytometry., Results: There were 102 SNPs associated with MRD in both cohorts (median odds ratio, 2.18; P < or = .0125), including 5 SNPs in the interleukin 15 (IL15) gene. Of these 102 SNPs, 21 were also associated with hematologic relapse (P < .05). Of 102 SNPs, 21 were also associated with antileukemic drug disposition, generally linking MRD eradication with greater drug exposure. In total, 63 of 102 SNPs were associated with early response, relapse, or drug disposition., Conclusion: Host genetic variations are associated with treatment response for childhood ALL, with polymorphisms related to leukemia cell biology and host drug disposition associated with lower risk of residual disease.

Adhesion of acute myeloid leukemia (AML) blasts in the bone marrow microenvironment confers protection from chemotherapy-induced apoptosis. One mechanism for retention of blasts within the bone marrow is adhesion via very late antigen-4 (VLA-4), the alpha(4)beta(1) integrin heterodimer that binds to its main ligands, fibronectin, and vascular cell adhesion molecule-1 (VCAM-1). To examine the relationship of functional expression of VLA-4 to prognosis in AML, we studied marrow samples from 175 adult AML patients who underwent induction chemotherapy with anthracycline and cytarabine on Southwest Oncology Group trials. The studies included flow cytometry and functional in vitro assays for ligand binding and maximal beta(1) activation. VLA-4 expression varied widely, with mean expression 60.6% for alpha(4), and was not significantly associated with response to chemotherapy, relapse-free, or overall survival (OS). However, increased binding of soluble VCAM-1 via VLA-4 was significantly associated with longer OS, corrected for age (P = .033). Estimated 5-year OS was 31% (95% confidence interval, 14%-48%) in 30 patients with soluble VCAM-1 binding greater than or equal to 40%, compared with 10% (confidence interval, 3%-17%) in 72 patients with lower binding. Adhesion and migratory properties of AML blasts thus appear to influence chemosensitivity and therefore may be therapeutic targets.

The underlying pathways that lead to relapse in childhood acute lymphoblastic leukemia (ALL) are unknown. To comprehensively characterize the molecular evolution of relapsed childhood B-precursor ALL, we used human 500K single-nucleotide polymorphism arrays to identify somatic copy number alterations (CNAs) in 20 diagnosis/relapse pairs relative to germ line. We identified 758 CNAs, 66.4% of which were less than 1 Mb, and deletions outnumbered amplifications by approximately 2.5:1. Although CNAs persisting from diagnosis to relapse were observed in all 20 cases, 17 patients exhibited differential CNA patterns from diagnosis to relapse. Of the 396 CNAs observed in 20 relapse samples, only 69 (17.4%) were novel (absent in the matched diagnosis samples). EBF1 and IKZF1 deletions were particularly frequent in this relapsed ALL cohort (25.0% and 35.0%, respectively), suggesting their role in disease recurrence. In addition, we noted concordance in global gene expression and DNA copy number changes (P = 2.2 x 10(-16)). Finally, relapse-specific focal deletion of MSH6 and, consequently, reduced gene expression were found in 2 of 20 cases. In an independent cohort of children with ALL, reduced expression of MSH6 was associated with resistance to mercaptopurine and prednisone, thereby providing a plausible mechanism by which this acquired deletion contributes to drug resistance at relapse.

Minimal residual disease (MRD) is an important predictor of relapse in acute lymphoblastic leukemia (ALL), but its relationship to other prognostic variables has not been fully assessed. The Children's Oncology Group studied the prognostic impact of MRD measured by flow cytometry in the peripheral blood at day 8, and in end-induction (day 29) and end-consolidation marrows in 2143 children with precursor B-cell ALL (B-ALL). The presence of MRD in day-8 blood and day-29 marrow MRD was associated with shorter event-free survival (EFS) in all risk groups; even patients with 0.01% to 0.1% day-29 MRD had poor outcome compared with patients negative for MRD patients (59% +/- 5% vs 88% +/- 1% 5-year EFS). Presence of good prognostic markers TEL-AML1 or trisomies of chromosomes 4 and 10 still provided additional prognostic information, but not in National Cancer Institute high-risk (NCI HR) patients who were MRD(+). The few patients with detectable MRD at end of consolidation fared especially poorly, with only a 43% plus or minus 7% 5-year EFS. Day-29 marrow MRD was the most important prognostic variable in multi-variate analysis. The 12% of patients with all favorable risk factors, including NCI risk group, genetics, and absence of days 8 and 29 MRD, had a 97% plus or minus 1% 5-year EFS with nonintensive therapy. These studies are registered at www.clinicaltrials.gov as NCT00005585, NCT00005596, and NCT00005603.

Chemotherapy resistance from imbalanced apoptosis regulation may contribute to poor outcome in leukaemias with t(4;11). Anti-apoptotic BCL-2 expression and target modulation were characterized in cell lines with t(4;11) and BCL-2 expression was examined in MLL and non-MLL infant/paediatric leukaemia cases by Western blot analysis and/or real-time polymerase chain reaction. Cytotoxicity of Genasensetrade mark (Oblimersen Sodium, G3139) alone or combined with cytotoxic drugs was assessed by MTT [(3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays of the cell lines, applying pharmacostatistical response surface modelling of drug interactions. Apoptosis and cell cycle were evaluated by flow cytometry in RS4:11 cells. Primary leukaemias and cell lines with t(4;11) expressed abundant BCL2 mRNA and protein. Variable, sometimes substantial BCL2 mRNA was detected in other leukaemia subtypes. G3139 reduced BCL2 mRNA and protein in RS4:11 cells. The most sensitive cell line to single-agent G3139 was RS4:11. Low G3139 concentrations sensitized RS4:11 and MV4-11 cells to select anti-leukaemia cytotoxic drugs. In RS4:11 cells, combining G3139 with doxorubicin (ADR) increased active caspase 3 and TUNEL staining compared to ADR alone, indicating greater apoptosis, and G3139 increased S-phase progression. The abundant BCL-2 affords a molecular target in leukaemias with t(4;11). G3139 exhibits preclinical activity and synergy with select cytotoxic agents in RS4:11 and MV4-11 cells, and these effects occur through apoptosis.

Gene expression profiling, the simultaneous measurement of the global patterns of gene expression in cells using microarray technologies, has held promise to improve diagnosis, risk classification, and outcome prediction in acute leukemias as well as in other human cancers. But how much has gene expression profiling impacted or improved current diagnosis and risk classification schemes and therapeutic targeting in acute myeloid leukemia (AML)? To date, gene expression profiling of AML has largely confirmed the presence of well-established recurring cytogenetic abnormalities, such as translocations, deletions, point mutations, or normal karyotypes, and has led to the identification of sets of genes reflective of these abnormalities. The use of expression profiling and statistical modeling to develop molecular classifiers that improve outcome prediction beyond traditional prognostic variables or the targeting of patients to specific therapies or transplantation has been less successful. Similarly, expression profiling has not led to the identification of many novel therapeutic targets in AML. Using advanced statistical modeling techniques, our group has focused on expression profiling in adult AML patients with poor risk features in order to identify novel cluster groups and potential targets for therapy in this highly resistant form of disease. To further advance the application of gene expression profiling and other comprehensive genomic and phosphoproteomic techniques to the study of AML, it would be ideal for the NCI Cooperative Groups to register all patients to common or intergroup collaborative clinical trials or registration studies in order to develop large, well-characterized cohorts of uniformly treated patients for future research studies.

Induction therapy for acute myeloid leukemia (AML) usually consists of 7 days of cytarabine at 100-200 mg/m(2)/day and an anthracycline. Such combinations produce complete response (CR) rates of 60-80% in patients with de novo AML. On the basis of a previous report, suggesting a higher CR rate using a regimen of standard daunomycin and cytarabine followed by 3 days of high-dose cytarabine (HDAC), 101 eligible patients received this regimen in a phase II trial. Sixty patients [59%, 95% confidence interval (CI) 49-69%] achieved a CR, and 10 patients died of infection during induction. Although cytogenetic risk group affected overall survival (P = 0.0016) and relapse-free survival (P = 0.0043), it had no impact on CR rate (P = 0.63). Patients received postremission therapy with repetitive courses of alternate day high-dose cytarabine; this was associated with considerable toxicity and the majority of patients could not receive all of the scheduled postremission therapy. The estimated median survival was 23 months (95% CI 15-34 months), and the estimated probability of surviving 5 years was 34% (95% CI 24-43%). The results of this intensive induction regimen were similar to that seen in previous trials and were not as promising as reported in the previous pilot study.

Repair of damage to DNA resulting from chemotherapy may influence drug toxicity and survival in response to treatment. We evaluated the role of polymorphisms in DNA repair genes APE1, XRCC1, ERCC1, XPD, and XRCC3 in predicting therapeutic outcomes of older adults with acute myeloid leukemia (AML) from 2 Southwest Oncology Group (SWOG) clinical trials. All patients received standard chemotherapy induction regimens. Using logistic and proportional hazards regression models, relationships between genotypes, haplotypes, and toxicities, response to induction therapy, and overall survival were evaluated. Patients with XPD Gln751C/Asp312G ('D') haplotype were more likely to have complete response (OR = 3.06; 95% CI, 1.44-6.70) and less likely to have resistant disease (OR = 0.32; 95%CI, 0.14-0.72) than patients with other haplotypes. ERCC1 polymorphisms were significantly associated with lung (P = .037) and metabolic (P = .041) toxicities, and patients with the XRCC3 241Met variant had reduced risk of liver toxicity (OR = 0.32; 95%CI, 0.11-0.95). Significant associations with other toxicities were also found for variant XPD genotypes/haplotypes. These data from clinical trials of older patients treated for AML indicate that variants in DNA repair pathways may have an impact on both outcomes of patients and toxicities associated with treatments. With validation of results in larger samples, these findings could lead to optimizing individual chemotherapy options.

We compared the gene expression profile of adult acute lymphoblastic leukemia (ALL) to normal hematopoietic and non-ALL samples using oligonucleotide arrays. Connective tissue growth factor (CTGF) was the highest overexpressed gene in B-cell ALL compared with the other groups, and displayed heterogeneous expression, suggesting it might have prognostic relevance. CTGF expression was examined by quantitative reverse transcriptase-polymerase chain reaction (ORT-PCR) on 79 adult ALL specimens. CTGF expression levels were significantly increased in ALL cases with B-lineage (P < .001), unfavorable cytogenetics (P < .001), and blasts expressing CD34 (P < .001). In a multivariate proportional hazards model, higher CTGF expression levels corresponded to worsening of overall survival (OS; hazard ratio 1.36, for each 10-fold increase in expression; P = .019). Further studies are ongoing to confirm the prognostic value of CTGF expression in ALL and to investigate its role in normal and abnormal lymphocyte biology.

The interpretation of low-level or non-reproducible amplification results in clinical quantitative polymerase chain reaction (Q-PCR) assays can be difficult to definitively resolve. Concerning minimal residual disease detection in leukaemia, indeterminate low-level results might create prognostic or therapeutic dilemmas. We evaluated low-level, ambiguous Q-PCR results in a study of paired diagnostic and end-induction (day 29) TEL-AML1 positive acute lymphoblastic leukaemia samples utilising a novel fluorescent primer ligation detection assay. The data presented here indicate that a significant number of low-level apparent Q-PCR positive results may be spurious or non-specific in nature, requiring additional technical manoeuvres for confirmation of true positive cases.

The t(8;21)(q22;q22) translocation is one of the most common genetic abnormalities in acute myeloid leukemia (AML), identified in 15% of all cases of AML, including 40-50% of FAB M2 subtype and rare cases of M0, M1 and M4 subtypes. The most commonly known AML1-ETO fusion protein (full-length AML1-ETO) from this translocation has 752 amino acids and contains the N-terminal portion of RUNX1 (also known as AML1, CBFalpha2 or PEBP2alphaB), including its DNA binding domain, and almost the entire RUNX1T1 (also known as MTG8 or ETO) protein. Although alterations of gene expression and hematopoietic cell proliferation have been reported in the presence of AML1-ETO, its expression does not lead to the development of leukemia. Here, we report the identification of a previously unknown alternatively spliced isoform of the AML1-ETO transcript, AML1-ETO9a, that includes an extra exon, exon 9a, of the ETO gene. AML1-ETO9a encodes a C-terminally truncated AML1-ETO protein of 575 amino acids. Expression of AML1-ETO9a leads to rapid development of leukemia in a mouse retroviral transduction-transplantation model. More importantly, coexpression of AML1-ETO and AML1-ETO9a results in the substantially earlier onset of AML and blocks myeloid cell differentiation at a more immature stage. These results indicate that fusion proteins from alternatively spliced isoforms of a chromosomal translocation may work together to induce cancer development.

To determine whether gene expression profiling could improve risk classification and outcome prediction in older acute myeloid leukemia (AML) patients, expression profiles were obtained in pretreatment leukemic samples from 170 patients whose median age was 65 years. Unsupervised clustering methods were used to classify patients into 6 cluster groups (designated A to F) that varied significantly in rates of resistant disease (RD; P < .001), complete response (CR; P = .023), and disease-free survival (DFS; P = .023). Cluster A (n = 24), dominated by NPM1 mutations (78%), normal karyotypes (75%), and genes associated with signaling and apoptosis, had the best DFS (27%) and overall survival (OS; 25% at 5 years). Patients in clusters B (n = 22) and C (n = 31) had the worst OS (5% and 6%, respectively); cluster B was distinguished by the highest rate of RD (77%) and multidrug resistant gene expression (ABCG2, MDR1). Cluster D was characterized by a "proliferative" gene signature with the highest proportion of detectable cytogenetic abnormalities (76%; including 83% of all favorable and 34% of unfavorable karyotypes). Cluster F (n = 33) was dominated by monocytic leukemias (97% of cases), also showing increased NPM1 mutations (61%). These gene expression signatures provide insights into novel groups of AML not predicted by traditional studies that impact prognosis and potential therapy.

Outcome for children with childhood acute lymphoblastic leukemia (ALL) who relapse is poor. To gain insight into the mechanisms of relapse, we analyzed gene-expression profiles in 35 matched diagnosis/relapse pairs as well as 60 uniformly treated children at relapse using the Affymetrix platform. Matched-pair analyses revealed significant differences in the expression of genes involved in cell-cycle regulation, DNA repair, and apoptosis between diagnostic and early-relapse samples. Many of these pathways have been implicated in tumorigenesis previously and are attractive targets for intervention strategies. In contrast, no common pattern of changes was observed among late-relapse pairs. Early-relapse samples were more likely to be similar to their respective diagnostic sample while we noted greater divergence in gene-expression patterns among late-relapse pairs. Comparison of expression profiles of early- versus late-relapse samples indicated that early-relapse clones were characterized by overexpression of biologic pathways associated with cell-cycle regulation. These results suggest that early-relapse results from the emergence of a related clone, characterized by the up-regulation of genes mediating cell proliferation. In contrast, late relapse appears to be mediated by diverse pathways.

We conducted a retrospective analysis of 968 adults with acute myeloid leukemia (AML) on 5 recent Southwest Oncology Group trials to understand how the nature of AML changes with age. Older study patients with AML presented with poorer performance status, lower white blood cell counts, and a lower percentage of marrow blasts. Multidrug resistance was found in 33% of AMLs in patients younger than age 56 compared with 57% in patients older than 75. The percentage of patients with favorable cytogenetics dropped from 17% in those younger than age 56 to 4% in those older than 75. In contrast, the proportion of patients with unfavorable cytogenetics increased from 35% in those younger than age 56 to 51% in patients older than 75. Particularly striking were the increases in abnormalities of chromosomes 5, 7, and 17 among the elderly. The increased incidence of unfavorable cytogenetics contributed to their poorer outcome, and, within each cytogenetic risk group, treatment outcome deteriorated markedly with age. Finally, the combination of a poor performance status and advanced age identified a group of patients with a very high likelihood of dying within 30 days of initiating induction therapy. The distinct biology and clinical responses seen argue for age-specific assessments when evaluating therapies for AML.

The transcription factor CEBPA is crucial for normal myeloid differentiation. CEBPA gene mutations have been reported in patients with acute myeloid leukaemia. The inevitable evolution of chronic myeloid leukaemia (CML) in chronic phase (CP) to a fatal blast crisis (BC) is assumed to result from the acquisition of additional genetic changes in the leukaemic clone. Gain of CEBPA mutations might represent a key event causing the differentiation block observed in myeloid CML-BC, but not in CML-CP. Here, no CEBPA mutation in 95 CML-BC patients was found, suggesting a limited role, if any, of CEBPA mutations in this disorder.

FLT3 internal tandem duplications (FLT3/ITDs) in the juxtamembrane domain are found in approximately 25% of acute myeloid leukemia (AML) patients, ranging in size from 3 to hundreds of nucleotides. We examined whether the sizes of FLT3/ITDs were associated with clinical outcomes in 151 AML patients enrolled in Southwest Oncology Group studies: S9333 and S9500. FLT3/ITDs were identified in 32% of patients (median ITD size = 39 nucleotides; range, 15-153 nucleotides). The CR rates were 35%, 67%, and 52% for patients with large (>or= 40), small (< 40), and no ITDs, respectively (P = .19). Increasing ITD size was associated with decreasing OS (estimated 5-year OS: large = 13%, small = 26%, and no ITD = 21%, P = .072) and RFS (estimated 5-year RFS: large = 13%, small = 27%, and no ITD = 34%, P = .017). These studies suggest that ITD size may have prognostic significance.

Relapse of acute promyelocytic leukemia (APL) following all-trans retinoic acid (ATRA) therapy has been associated with the acquisition of mutations in the high-affinity ATRA binding site in PML-RARalpha, but little information is available about the selection dynamics of the mutation-harboring subclones. In this study, 6/18 patients treated with sequential ATRA and chemotherapy on protocol INT0129 relapsed with complete replacement of the nonmutant pretreatment APL cell population by a PML-RARalpha mutant subclone. Two patients relapsed in proximity of ATRA treatment; however, in four patients there was a 6-48 month hiatus between the last ATRA treatment and relapse. The mutant subclones were not detectable in samples tested > or = 3 months before relapse at > or = 1 in 10(2) (10(-2)) sensitivity. In one patient, a functionally weak mutation was detected at 10(-4) sensitivity before therapy but only limited pre-relapse enrichment of the mutant subclone was observed on subsequent ATRA therapy. These results indicate that proximate ATRA selection pressure is frequently not the main determinant for the emergence of strongly dominant PML-RARalpha mutant subclones and suggest that APL subclones harboring PML-RARalpha mutations are predisposed to the acquisition of secondary genetic/epigenetic alterations that result in a growth/survival advantage.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease with distinct biological and clinical features. The biologic basis of the stereotypical progression from chronic phase through accelerated phase to blast crisis is poorly understood. We used DNA microarrays to compare gene expression in 91 cases of CML in chronic (42 cases), accelerated (17 cases), and blast phases (32 cases). Three thousand genes were found to be significantly (P < 10(-10)) associated with phase of disease. A comparison of the gene signatures of chronic, accelerated, and blast phases suggest that the progression of chronic phase CML to advanced phase (accelerated and blast crisis) CML is a two-step rather than a three-step process, with new gene expression changes occurring early in accelerated phase before the accumulation of increased numbers of leukemia blast cells. Especially noteworthy and potentially significant in the progression program were the deregulation of the WNT/beta-catenin pathway, the decreased expression of Jun B and Fos, alternative kinase deregulation, such as Arg (Abl2), and an increased expression of PRAME. Studies of CML patients who relapsed after initially successful treatment with imatinib demonstrated a gene expression pattern closely related to advanced phase disease. These studies point to specific gene pathways that might be exploited for both prognostic indicators as well as new targets for therapy.

Deletion of the long arm of chromosome 9, del(9q), is a recurring chromosomal aberration in acute myeloid leukemia (AML) that is frequently associated with t(8;21). The critical gene products affected by del(9q) are unknown but likely cooperate with the AML1/ETO fusion gene created by t(8;21) in leukemogenesis. In 43 AML samples with del(9q), we used high-density microsatellite markers to define the commonly deleted region (CDR) to less than 2.4 Mb. We found no homozygous loss at any locus tested. The CDR contains 7 known genes, FRMD3, UBQLN1, GKAP42, KIF27, HNRPK, SLC28A3, and NTRK2, and 4 novel genes, RASEF, C9orf103, C9orf64, and C9orf76. In addition, TLE1 and TLE4 are adjacent to the CDR. We performed a comprehensive mutational analysis of the coding regions of all these genes. No sequence variations absent in normal controls were seen in more than a single del(9q) AML sample. Expression of 7 of the 10 genes examined was significantly down-regulated in del(19q)AML as compared with the CD34-purified progenitors from normal individuals, a pattern distinct from that seen in AML samples with a normal karyotype. The results of our studies are consistent with a model of tumor suppression mediated by haploinsufficiency of critical genes in del(9q) AML., ((c) 2005 Wiley-Liss, Inc.)

Core binding factor (CBF) participates in specification of the hematopoietic stem cell and functions as a critical regulator of hematopoiesis. Translocation or point mutation of acute myeloid leukemia 1 (AML1)/RUNX1, which encodes the DNA-binding subunit of CBF, plays a central role in the pathogenesis of acute myeloid leukemia and myelodysplasia. We characterized the t(X;21)(p22.3;q22.1) in a patient with myelodysplasia that fuses AML1 in-frame to the novel partner gene FOG2/ZFPM2. The reciprocal gene fusions AML1-FOG2 and FOG2-AML1 are both expressed. AML1-FOG2, which fuses the DNA-binding domain of AML1 to most of FOG2, represses the transcriptional activity of both CBF and GATA1. AML1-FOG2 retains a motif that recruits the corepressor C-terminal binding protein (CtBP) and these proteins associate in a protein complex. These results suggest a central role for CtBP in AML1-FOG2 transcriptional repression and implicate coordinated disruption of the AML1 and GATAdevelopmental programs in the pathogenesis of myelodysplasia.

Fms-like tyrosine kinase 3 (FLT3) mutations are associated with unfavorable outcomes in children with acute myeloid leukemia (AML). We used DNA microarrays to identify gene expression profiles related to FLT3 status and outcome in childhood AML. Among 81 diagnostic specimens, 36 had FLT3 mutations (FLT3-MUs), 24 with internal tandem duplications (ITDs) and 12 with activating loop mutations (ALMs). In addition, 8 of 19 specimens from patients with relapses had FLT3-MUs. Predictive analysis of microarrays (PAM) identified genes that differentiated FLT3-ITD from FLT3-ALM and FLT3 wild-type (FLT3-WT) cases. Among the 42 specimens with FLT3-MUs, PAM identified 128 genes that correlated with clinical outcome. Event-free survival (EFS) in FLT3-MU patients with a favorable signature was 45% versus 5% for those with an unfavorable signature (P = .018). Among FLT3-MU specimens, high expression of the RUNX3 gene and low expression of the ATRX gene were associated with inferior outcome. The ratio of RUNX3 to ATRX expression was used to classify FLT3-MU cases into 3 EFS groups: 70%, 37%, and 0% for low, intermediate, and high ratios, respectively (P < .0001). Thus, gene expression profiling identified AML patients with divergent prognoses within the FLT3-MU group, and the RUNX3 to ATRX expression ratio should be a useful prognostic indicator in these patients.

Cholesterol levels are abnormally increased in many acute myeloid leukemia (AML) samples exposed in vitro to chemotherapy. Blocking these acute cholesterol responses selectively sensitizes AML cells to therapeutics. Thus, defining the molecular mechanisms by which AML cells accomplish these protective cholesterol increments might elucidate novel therapeutic targets. We now report that the levels of mRNAs encoding the cholesterol synthesis-regulating enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and the cholesterol-importing low-density lipoprotein (LDL) receptor were both increased by daunorubicin (DNR) or cytarabine (ARA-C) treatments in almost three fourths of cultured AML samples. However, less than one third of AML samples significantly increased LDL accumulation during drug treatments, suggesting that de novo synthesis is the primary mechanism by which most AML cells increase cholesterol levels during drug exposures. LDL increments were not correlated with cholesterol increments in ARA-C-treated AML samples. However, LDL and cholesterol increments did correlate in DNR-treated AML samples where they were measured, suggesting that a subset of AMLs may rely on increased LDL accumulation during treatment with particular drugs. Our data suggest that cholesterol synthesis inhibitors may improve the efficacy of standard antileukemia regimens, but that for maximum benefit, therapy may need to be tailored for individual patients with leukemia.

A new human IL-2 dependent leukemic cell line with a natural killer (NK) cell phenotype, IMC-1, was established from an adult patient with aggressive NK cell leukemia. The IMC-1 cell line expresses the CD56, CD2, CD11a, CD38 and HLA-DR cell surface antigens, whereas the CD16 and CD8 antigens expressed on the primary leukemic blasts from which the cell line was derived were lost after 7 and 28 weeks of culture, respectively. The IMC-1 cell line displays functional NK cytotoxicity and lyses target cells in a non-MHC restricted, antibody-independent manner with equal or superior efficiency to freshly isolated NK cells. Cytogenetic analysis at presentation and after 55 weeks in culture revealed complex structural and numerical abnormalities, defined by classic G-banding and by spectral karyotyping (SKY). Three apparently intact copies of chromosome 8 occurred in the diagnostic bone marrow specimen; the cell line also contains three copies of chromosome 8 but each was structurally altered. The development and detailed characterization of this new NK leukemic cell line will facilitate biologic and functional studies of NK cells and chromosomal aberrations potentially important in leukemic transformation.